A kit for inducing differentiation of stem cells into megakaryocytes and / or platelets and uses thereof

Abstract

The application belongs to the technical field of biology, and discloses a kit for inducing stem cells to differentiate into megakaryocytes and / or platelets and application thereof. The kit can induce stem cells to differentiate into megakaryocytes and / or platelets without introducing exogenous cells and exogenous genes. The kit can highly simulate the generation process of megakaryocytes and platelets in vivo, and can induce stem cells into mesendoderm cells, hematopoietic endothelial cells and megakaryocyte-erythroid progenitor cells in sequence. The megakaryocyte-erythroid progenitor cells further develop into megakaryocytes with a multiploid characteristic, and finally produce stem cell-derived platelets with similar functions to natural platelets. The stem cells have the potential for unlimited proliferation, and can be massively expanded in vitro, which provides an unlimited cell source for the in vitro preparation of platelets.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a kit for inducing stem cells to differentiate into megakaryocytes and / or platelets and application thereof. BACKGROUND

[0002] Platelets are small pieces of cytoplasm that are shed from mature multinucleated megakaryocytes in the bone marrow after cytoskeleton rearrangement and cytoplasmic cleavage, and can survive for 5-7 days in the blood circulation. In the human body, the main role of platelets is to act as a regulator of hemostasis and thrombosis; in addition, platelets have important functions in the repair of damaged blood vessels and the protection of vascular endothelium; at the same time, studies have shown that platelets also play an important role in innate immunity, regulation of tumor growth and extravascular leakage. Normal human body contains 10-30 billion platelets per liter of blood. In clinical practice, many diseases, such as aplastic anemia, leukemia and systemic lupus erythematosus, can cause a decrease in the number of platelets. When the number of platelets in the human body per liter of blood is less than 10-20 billion, the hemostatic system will be severely impaired, with a risk of spontaneous bleeding, and platelet injection is required for treatment. At present, platelets used for clinical treatment mainly come from blood donors, and at least 25 billion platelets are needed for a platelet treatment unit, which needs to be extracted from 1 liter of whole blood, equivalent to 5 blood donors to complete; moreover, since the survival time of platelets in the body is only about 1 week, the drug efficacy period of platelet treatment is limited. For patients with long-term insufficient platelet content, repeated platelet injection is needed to maintain the therapeutic effect. The limited source of platelets and the great demand will undoubtedly cause serious medical pressure. Therefore, it is urgent to find a way to obtain platelets other than blood donation.

[0003] Megakaryocyte development in vivo is a complex process. First, hematopoietic stem cells at the top of the hematopoietic lineage development undergo asymmetric division in the bone marrow to form two cells, one of which is a hematopoietic stem cell that maintains self-renewal ability, and the other is a hematopoietic progenitor cell that can develop into multiple hematopoietic lineages but lacks self-renewal ability. Subsequently, hematopoietic progenitor cells are induced by cytokines and regulated by specific transcription factors to differentiate into myeloid progenitor cells or lymphoid progenitor cells. When myeloid progenitor cells are produced, they can continue to develop and produce megakaryocyte-erythroid progenitor cells with the potential to differentiate into both erythroid and megakaryocyte lineages. Megakaryocyte-erythroid progenitor cells can develop into the erythroid lineage under the regulation of factors such as EPO and KLF1, and can develop into the megakaryocyte lineage under the regulation of factors such as TPO and FLI1, and migrate to the location near the bone marrow sinusoid. Early differentiating megakaryocytes first express two specific immunomarkers, CD61 and CD41a. As megakaryocytes develop and mature, surface antigens CD42a, CD42b, and CD42d begin to be expressed, and the expression level reaches the highest at the mature stage; the expression of CD42b may gradually be lost with the production of platelets. Through transcriptomic analysis of megakaryocytes in vivo, it was found that there are at least three different types of mature megakaryocytes in the human body, including megakaryocytes that support the maintenance of hematopoietic stem cells, megakaryocytes with immune response function, and megakaryocytes with platelet production capacity. One characteristic of platelet-producing megakaryocytes is that they can undergo a unique endomitosis during the development and maturation stage, and DNA can be replicated to form polyploidy, but the cytoplasm cannot be divided, resulting in the formation of polyploid cells in mature platelet-producing megakaryocytes. Mature polyploid megakaryocytes have higher protein synthesis capacity, providing sufficient material basis for platelet production.

[0004] Currently, various cells can be used for the biological manufacture of platelets, including hematopoietic stem cells isolated from bone marrow, peripheral blood, and umbilical cord blood, as well as pluripotent stem cells. Compared with pluripotent stem cells, hematopoietic stem cells, as precursor cells of platelets, have a more direct and rapid "pathway" to differentiate into megakaryocytes and platelets. Hematopoietic stem cells can be isolated from bone marrow, peripheral blood, and umbilical cord blood. The proliferation potential, differentiation ability to megakaryocyte lineage, and intrinsic ability to produce platelets of hematopoietic stem cells from different sources are different. Compared with umbilical cord blood hematopoietic stem cells, bone marrow and peripheral blood-derived hematopoietic stem cells have higher polyploidy levels and higher platelet production potential. However, the source and storage of umbilical cord blood are superior to the former two, so umbilical cord blood-derived hematopoietic stem cells have stronger competitiveness, and multiple teams have been committed to the research of hematopoietic stem cell differentiation into platelets in vitro and have established the corresponding culture system.

[0005] Human pluripotent stem cells (hPSCs) possess multipotent differentiation potential, and large-scale production of mature platelets derived from hPSCs is a potential alternative to meet clinical platelet needs. Several pathways for in vitro platelet production based on hPSCs have been proposed, including co-culturing hESCs with stromal cells to differentiate into megakaryocytes and generate platelets. [1] Gene editing was used to induce the overexpression of megakaryocyte lineage-related specific transcription factors, such as GATA1, FLI1, and TAL1, in human induced pluripotent stem cells (hiPSCs) to induce the production of megakaryocytes and platelets. [2] ; and the use of specific combinations of cytokines to directionally induce the production of cells at various stages of development and ultimately obtain megakaryocytes to produce platelets, etc. [3] .

[0006] hPSCs were initially induced into megakaryocytes and platelets in vitro by combining hESCs with OP9 stromal cells. [3] Achieved through co-culture with mouse embryonic mesenchymal cells [1] Subsequent studies have shown that megakaryocytes and platelets can be generated from multiple hESC lineages under serum-free and nutrient-free conditions. [4] This method uses hESC-induced hemangioblasts as cellular intermediates to generate platelets with platelet ultrastructure and morphological characteristics. In vitro, these platelets can be activated by thrombin, and when transplanted in vivo, they can integrate into the blood vessel at the laser-induced vascular injury site to form thrombi. [4] .

[0007] Recently, researchers have used hiPSC differentiation [5,6] and reprogramming technology [7,8] Megakaryocytes and platelets were generated in vitro. Researchers in Koji Eto's lab used overexpression of BMI1 and BCL-XL to inhibit cell senescence and apoptosis in hiPSC-derived hematopoietic progenitor cells, and overexpressed c-MYC to promote cell proliferation during the differentiation of hematopoietic progenitor cells into megakaryocyte progenitor cells, thereby establishing stable immortalized megakaryocyte progenitor cells. [9] The immortalized megakaryocyte progenitor cells, when the overexpression of BMI1, BCL-XL, and c-MYC was turned off during subsequent differentiation, were able to develop normally into mature megakaryocytes and produce functional platelets. Furthermore, researchers in Cedric Ghevaert's team promoted the differentiation of hiPSCs into megakaryocytes and platelets in a feederless culture system by overexpressing three megakaryocyte-specific transcription factors: GATA1, FLI1, and TAL1, thereby improving differentiation efficiency. [2]In addition, researchers have reprogrammed fibroblasts into megakaryocytes using three transcription factors NEF2, MAFG and MAFK, and the megakaryocytes generated by injection into immunodeficient mice can produce functional platelets [7] Similarly, by introducing six transcription factors GATA2, RUNX1, GATA1, TAL1, LMO2 and c-MYC, fibroblasts can be successfully converted into megakaryocyte progenitor cells

[10] .

[0008] Although the method of using natural hematopoietic stem cells to differentiate into megakaryocytes and produce platelets in vitro has certain advantages, it also has some disadvantages. For example, hematopoietic stem cells are mainly derived from blood donors, and blood donation resources are limited. Secondly, the number of hematopoietic stem cells that can be obtained from umbilical cord blood, bone marrow or peripheral blood is very limited, so it is often necessary to first expand the hematopoietic stem cells collected in vitro to increase the number of hematopoietic stem cells before megakaryocyte differentiation. However, hematopoietic stem cells often undergo non-directional differentiation during this expansion dependent on specific cytokine combinations, which will lead to a decrease in the efficiency and yield of hematopoietic stem cell differentiation into megakaryocytes. More importantly, the use of hematopoietic stem cells to prepare platelets in vitro cannot truly meet the clinical demand for platelets. In addition, the use of co-culture with stromal cells to induce hPSCs to differentiate into megakaryocytes has the risk of introducing exogenous cells, which greatly limits the use of the generated platelets in clinical practice. Secondly, although the megakaryocytes generated by gene editing through the introduction of exogenous genes have certain advantages in efficiency, the products obtained still have certain potential biological safety risks. Therefore, the use of platelets generated by this method in clinical practice will also be limited.

[0009] [1] Takayama N, Nishikii H, Usui J, et al. Generation of functional platelets from human embryonic stem cells in vitro via ES-sacs, VEGF-promoted structures that concentrate hematopoietic progenitors [J]. Blood, 2008, 111(11): 5298-306.

[0010] [2] Moreau T, Evans A L, Vasquez L, et al. Large-scale production of megakaryocytes from human pluripotent stem cells by chemically defined forward programming [J]. Nat Commun, 2016, 7: 11208.

[0011] [3] Zhang B, Wu X, Zi G, et al. Large-scale generation of megakaryocytes from human embryonic stem cells using transgene-free and stepwise defined suspension culture conditions [J]. Cell Prolif, 2021, 54(4): e13002.

[0012] [4] Lu S J, Li F, Yin H, et al. Platelets generated from human embryonic stem cells are functional in vitro and in the microcirculation of living mice [J]. Cell Res, 2011, 21(3): 530-45.

[0013] [5] Nakagawa Y, Nakamura S, Nakajima M, et al. Two differential flows in a bioreactor promoted platelet generation from human pluripotent stem cell-derived megakaryocytes [J]. Exp Hematol, 2013, 41(8): 742-8.

[0014] [6] Takayama N, Nishimura S, Nakamura S, et al. Transient activation of c-MYC expression is critical for efficient platelet generation from human induced pluripotent stem cells [J]. J Exp Med, 2010, 207(13): 2817-30.

[0015] [7] Ono Y, Wang Y, Suzuki H, et al. Induction of functional platelets from mouse and human fibroblasts by p45NF-E2 / Maf [J]. Blood, 2012, 120(18): 3812-21.

[0016] [8] Masuda S, Li M, Izpisua Belmonte J C. In vitro generation of platelets through direct conversion: first report in My Knowledge (iMK) [J]. Cell Res, 2013, 23(2): 176-8.

[0017] [9] Nakamura S, Takayama N, Hirata S, et al. Expandable megakaryocyte cell lines enable clinically applicable generation of platelets from human induced pluripotent stem cells [J]. Cell Stem Cell, 2014, 14(4): 535-48.

[0018]

[10] Pulecio J, Alejo-Valle O, Capellera-Garcia S, et al. Direct conversion of fibroblasts to megakaryocyte progenitors [J]. Cell Rep, 2016, 17(3): 671-683. SUMMARY

[0019] The first aspect of the present application aims to provide a kit for inducing stem cells to differentiate into megakaryocytes.

[0020] The second aspect of the present application aims to provide a kit for inducing stem cells to differentiate into platelets.

[0021] The third aspect of the present application aims to provide the use of the kits of the first and second aspects.

[0022] The fourth aspect of the present application aims to provide a method for inducing stem cells to differentiate into megakaryocytes and / or platelets.

[0023] The fifth aspect of the present application aims to provide a megakaryocyte.

[0024] The sixth aspect of the present application aims to provide a platelet.

[0025] The seventh aspect of the present application aims to provide the use of the platelet of the sixth aspect of the present application.

[0026] The eighth aspect of the present application aims to provide a medicament.

[0027] In order to achieve the above-mentioned objects, the technical solutions adopted by the present application are as follows:

[0028] The first aspect of the present application provides a kit for inducing stem cells to differentiate into megakaryocytes, comprising: a fourth culture medium; the fourth culture medium is a basic culture medium comprising at least one, at least two or at least three of the following: growth factors, colony stimulating factors, TGFβ / ALK inhibitors.

[0029] Preferably, the fourth culture medium is a basic culture medium comprising growth factors, colony stimulating factors and TGFβ / ALK inhibitors.

[0030] Preferably, the growth factors of the fourth culture medium comprise at least one of epidermal growth factor (EGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), hepatocyte growth factor (HGF), insulin-like growth factor-I (IGF-1), IGF-Ⅱ, leukemia inhibitory factor (LIF), nerve growth factor (NGF), oncostatin M (OSM), platelet-derived endothelial cell growth factor (PDECGF), transforming growth factor-α (TGF-α), vascular endothelial cell growth factor (VEGF); further preferably, the growth factors of the fourth culture medium comprise at least one of fibroblast growth factor (FGF) and vascular endothelial cell growth factor (VEGF); and more preferably, the growth factors of the fourth culture medium comprise fibroblast growth factor (FGF) and vascular endothelial cell growth factor (VEGF).

[0031] Preferably, the fibroblast growth factor comprises at least one of acidic fibroblast growth factor, basic fibroblast growth factor; further preferably, the fibroblast growth factor comprises basic fibroblast growth factor.

[0032] Preferably, the colony stimulating factor of the fourth medium comprises at least one of G-CSF (granulocyte colony stimulating factor), M-CSF (macrophage colony stimulating factor), GM-CSF (recombinant human granulocyte-macrophage colony stimulating factor), multi-CSF (multiple colony stimulating factor, also known as IL-3), EPO (erythropoietin), TPO, SCF, FlT3L; further preferably, the colony stimulating factor of the fourth medium comprises EPO.

[0033] Preferably, the TGFβ / ALK inhibitor of the fourth medium comprises at least one of SB431542, SB-505, A-83-01, GW6604, IN-1130, Ki26894, LY2157299, LY364947 (HTS-466284), LY550410, LY573636, LY580276, NPC-30345, SB-505124, SD-093, Sm16, SM305, SX-007, Antp-Sm2A, LY2109761; further preferably, the TGFβ / ALK inhibitor of the fourth medium comprises SB431542.

[0034] Preferably, the concentration of the growth factor in the fourth medium is 20-150 ng / mL; further 60-90 ng / mL; more further 70 ng / mL.

[0035] Preferably, the concentration of the vascular endothelial growth factor in the fourth medium is 10-100 ng / mL; further 40-60 ng / mL; more further 50 ng / mL.

[0036] Preferably, the concentration of the basic fibroblast growth factor in the fourth medium is 10-50 ng / mL; further 20-30 ng / mL; more further 20 ng / mL.

[0037] Preferably, the concentration of the colony stimulating factor in the fourth medium is 1-10 U / mL; further 1-5 U / mL; more further 3 U / mL.

[0038] Preferably, the concentration of the TGFβ / ALK inhibitor in the fourth medium is 1-20 μM; further 1-5 μM; more further 4 μM.

[0039] Preferably, the base medium of the fourth medium comprises: IMDM (Iscove's Modified Dulbecco's Medium), DMEM medium, Ham's F-12 medium, STEMdiff TM APEL TM 2 medium; further preferably, the base medium of the fourth medium comprises STEMdiff TM APEL TM 2 medium.

[0040] In the present application, the fourth medium is used to induce hematopoietic endothelial cells (early hematopoietic cells) to differentiate into megakaryocytic erythroid progenitor cells.

[0041] Preferably, the kit for inducing stem cells to differentiate into megakaryocytes further comprises: a sixth medium, which is a base medium comprising at least one or at least two of: colony stimulating factor, interleukin.

[0042] Preferably, the sixth medium is a base medium comprising colony stimulating factor and interleukin.

[0043] Preferably, the colony stimulating factor of the sixth medium comprises at least two of: G-CSF (granulocyte colony-stimulating factor), M-CSF (macrophage colony-stimulating factor), GM-CSF (recombinant human granulocyte-macrophage colony-stimulating factor), multi-CSF (multiple colony-stimulating factor, also known as IL-3), EPO (erythropoietin), TPO, SCF, FlT3L; further preferably, the colony stimulating factor of the sixth medium comprises SCF, TPO and IL3.

[0044] Preferably, the interleukin of the sixth medium comprises at least two of: IL-1, IL-2, IL-6, IL-7, IL-11, IL-15, IL-18, IL-21, IL-27; further preferably, the interleukin of the sixth medium comprises IL-6 and IL-11.

[0045] Preferably, the concentration of the colony stimulating factor in the sixth medium is 70-450 ng / mL; further 145-255 ng / mL; and more further 170 ng / mL.

[0046] Preferably, the concentration of the SCF in the sixth medium is 10-150 ng / mL; further 50-100 ng / mL; and more further 50 ng / mL.

[0047] Preferably, the concentration of the TPO in the sixth medium is 50-250 ng / mL; further 75-125 ng / mL; and more further 100 ng / mL.

[0048] Preferably, the concentration of the IL3 in the sixth medium is 10-50 ng / mL; further 20-30 ng / mL; and more further 20 ng / mL.

[0049] Preferably, the concentration of the interleukin in the sixth medium is 20-140 ng / mL; further 60-90 ng / mL; and more further 70 ng / mL.

[0050] Preferably, the concentration of the IL6 in the sixth medium is 10-100 ng / mL; further 40-60 ng / mL; and more further 50 ng / mL.

[0051] Preferably, the concentration of the IL11 in the sixth medium is 10-40 ng / mL; further 20-30 ng / mL; and more further 20 ng / mL.

[0052] Preferably, the base medium of the sixth medium comprises at least one of IMDM (Iscove's Modified Dulbecco's Medium), DMEM medium, Ham's F-12 medium, STEMdiff TM APEL TM 2 medium, StemSpan TM SFEM II medium; and further preferably, the base medium of the sixth medium comprises StemSpan TM SFEM II medium.

[0053] In the present application, the sixth medium is used for inducing megakaryoerythroid progenitor cells to differentiate into mature megakaryocytes.

[0054] Preferably, the kit for inducing stem cells to differentiate into megakaryocytes further comprises: a second medium and a third medium, the second medium and the third medium being base media comprising growth factors;

[0055] The growth factors of the second culture medium comprise any one of epidermal growth factor (EGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), hepatocyte growth factor (HGF), insulin-like growth factor-I (IGF-1), IGF-II, leukemia inhibitory factor (LIF), nerve growth factor (NGF), oncostatin M (OSM), platelet-derived endothelial cell growth factor (PDECGF), transforming growth factor-α (TGF-α), and vascular endothelial cell growth factor (VEGF).

[0056] The growth factors of the third culture medium comprise at least two of epidermal growth factor (EGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), hepatocyte growth factor (HGF), insulin-like growth factor-I (IGF-1), IGF-II, leukemia inhibitory factor (LIF), nerve growth factor (NGF), oncostatin M (OSM), platelet-derived endothelial cell growth factor (PDECGF), transforming growth factor-α (TGF-α), and vascular endothelial cell growth factor (VEGF).

[0057] Preferably, the growth factors of the second culture medium comprise vascular endothelial cell growth factor (VEGF).

[0058] Preferably, the growth factors of the third culture medium comprise fibroblast growth factor (FGF) and vascular endothelial cell growth factor (VEGF).

[0059] Preferably, the fibroblast growth factor comprises at least one of acidic fibroblast growth factor and basic fibroblast growth factor; further preferably, the fibroblast growth factor comprises basic fibroblast growth factor.

[0060] Preferably, the concentration of the growth factor in the second culture medium is 10-100 ng / mL; further 40-60 ng / mL; and more further 40 ng / mL.

[0061] Preferably, the concentration of the growth factor in the third culture medium is 20-150 ng / mL; further 50-70 ng / mL; and more further 60 ng / mL.

[0062] Preferably, the concentration of the vascular endothelial growth factor in the third culture medium is 10-100 ng / mL; further 40-50 ng / mL; and more further 40 ng / mL.

[0063] Preferably, the concentration of the basic fibroblast growth factor in the third culture medium is 10-50 ng / mL; further 10-20 ng / mL; more further 20 ng / mL.

[0064] Preferably, the base medium of the second culture medium comprises at least one of IMDM (Iscove's Modified Dulbecco's Medium) medium, DMEM medium, Ham's F-12 medium, STEMdiff TM APEL TM 2 medium; further preferably, the base medium of the second culture medium comprises STEMdiff TM APEL TM 2 medium.

[0065] Preferably, the base medium of the third culture medium comprises at least one of IMDM (Iscove's Modified Dulbecco's Medium) medium, DMEM medium, Ham's F-12 medium, STEMdiff TM APEL TM 2 medium; further preferably, the base medium of the third culture medium comprises STEMdiff TM APEL TM 2 medium.

[0066] In the present application, the second culture medium and the third culture medium are used to induce mesendoderm cells to differentiate into hematopoietic endothelial cells (early hematopoietic cells).

[0067] Preferably, the kit for inducing stem cells to differentiate into megakaryocytes further comprises: a first culture medium, the first culture medium being a base medium comprising at least one, at least two, at least three, or at least four of the following: a ROCK inhibitor, a BMP signaling pathway activator, a GSK3 inhibitor, Activin A.

[0068] Preferably, the ROCK inhibitor of the first culture medium comprises at least one of Blebbistatin, HA-100, Y-27632, HA-1077, KD-025, Y-33075, Narciclasine; further preferably, the ROCK inhibitor of the first culture medium comprises Y-27632.

[0069] Preferably, the activator of BMP signaling pathway of the first medium comprises at least one of BMP2, BMP4, SB4, SJ000291942, SJ000063181, SJ000370178, isoliquiritigenin, dihydroxypropyl resorcinol, apigenin, and biochanin A; further preferably, the activator of BMP signaling pathway of the first medium comprises BMP4.

[0070] Preferably, the GSK-3 inhibitor of the first medium comprises at least one of GSK-3a inhibitor, GSK-3b inhibitor; further preferably, the GSK-3 inhibitor of the first medium comprises at least one of B216763, TWS119, NP031112, SB216763, CHIR-98014, AZD2858, AZD1080, SB415286, LY2090314, CHIR-99021; more further preferably, the GSK-3 inhibitor of the first medium comprises CHIR-99021.

[0071] Preferably, the concentration of the ROCK inhibitor in the first medium is 1-20 µM; further 5-15 µM; more further 10 µM.

[0072] Preferably, the concentration of the activator of BMP signaling pathway in the first medium is 5-50 ng / mL; further 5-25 ng / mL; more further 10 ng / mL.

[0073] Preferably, the concentration of the GSK3 inhibitor in the first medium is 0.5-5 µM; further 1-4 µM; more further 3 µM.

[0074] Preferably, the concentration of the Activin A in the first medium is 1-20 ng / mL; further 1-5 ng / mL; more further 2 ng / mL.

[0075] Preferably, the basal medium of the first medium comprises at least one of IMDM (Iscove's Modified Dulbecco's Medium), DMEM medium, Ham's F-12 medium, STEMdiff TM APEL TM 2 medium; further preferably, the basal medium of the first medium comprises STEMdiff TM APEL TM 2 medium.

[0076] In the present application, the first medium is used for inducing stem cells to differentiate into mesendoderm cells.

[0077] Preferably, the kit for inducing stem cells to differentiate into megakaryocytes further comprises: a fifth medium, which is a basic medium comprising at least one or at least two of: a colony stimulating factor, an interleukin.

[0078] Preferably, the fifth medium is a basic medium comprising a colony stimulating factor and an interleukin.

[0079] Preferably, the colony stimulating factor of the fifth medium comprises at least two of: G-CSF (granulocyte colony stimulating factor), M-CSF (macrophage colony stimulating factor), GM-CSF (recombinant human granulocyte-macrophage colony stimulating factor), multi-CSF (multiple colony stimulating factor, also known as IL-3), EPO (erythropoietin), TPO, SCF, FlT3L; further preferably, the colony stimulating factor of the fifth medium comprises SCF, TPO and IL3.

[0080] Preferably, the interleukin of the fifth medium comprises at least two of: IL-1, IL-2, IL-6, IL-7, IL-11, IL-15, IL-18, IL-21, IL-27; further preferably, the interleukin of the fifth medium comprises IL-6 and IL-11.

[0081] Preferably, the concentration of the colony stimulating factor in the fifth medium is 85-350 ng / mL; further 85-170 ng / mL; more further 170 ng / mL.

[0082] Preferably, the concentration of the SCF in the fifth medium is 25-100 ng / mL; further 25-50 ng / mL; more further 50 ng / mL.

[0083] Preferably, the concentration of the TPO in the fifth medium is 50-200 ng / mL; further 50-100 ng / mL; more further 100 ng / mL.

[0084] Preferably, the concentration of the IL3 in the fifth medium is 10-50 ng / mL; further XX-XX ng / mL; more further 20 ng / mL.

[0085] Preferably, the concentration of the interleukin in the fifth medium is 60-200 ng / mL; further 60-85 ng / mL; more further 70 ng / mL.

[0086] Preferably, the concentration of IL6 in the fifth medium is 50-150 ng / mL; further 50-75 ng / mL; more further 50 ng / mL.

[0087] Preferably, the concentration of IL11 in the fifth medium is 10-50 ng / mL; further 10-20 ng / mL; more further 20 ng / mL.

[0088] Preferably, the basal medium of the fifth medium comprises at least one of IMDM (Iscove's Modified Dulbecco's Medium), DMEM medium, Ham's F-12 medium, STEMdiff TM APEL TM 2 medium, StemSpan TM SFEM II medium; further preferably, the basal medium of the fifth medium comprises StemSpan TM SFEM II medium.

[0089] In the present application, the fifth medium is used for the maintenance culture of megakaryocytic erythroid progenitor cells.

[0090] Preferably, the stem cell is a stem cell with multi-differentiation potential.

[0091] Preferably, the stem cell with multi-differentiation potential comprises at least one of embryonic stem cell, parthenogenetic stem cell, induced pluripotent stem cell, mesenchymal stem cell, adipose stem cell, cord blood stem cell; further preferably, the stem cell with multi-differentiation potential comprises embryonic stem cell.

[0092] Preferably, the stem cell is derived from mammal; further derived from primate, more further derived from human.

[0093] Preferably, the embryonic stem cell is a commercialized human embryonic stem cell line.

[0094] Preferably, the embryonic stem cell is a stem cell isolated or obtained from human embryo which has not developed in vivo for 14 days after fertilization.

[0095] Preferably, the megakaryocyte is a mature megakaryocyte.

[0096] In the second aspect of the present application, a kit for inducing stem cell to differentiate into platelet is provided, comprising the kit of the first aspect of the present application.

[0097] Preferably, the kit further comprises: a seventh medium; the seventh medium is a basal medium comprising at least one or at least two of: a colony stimulating factor, an interleukin.

[0098] Preferably, the seventh medium is a basal medium comprising a colony stimulating factor and an interleukin.

[0099] Preferably, the colony stimulating factor of the seventh medium comprises any one of: G-CSF (granulocyte colony-stimulating factor), M-CSF (macrophage colony-stimulating factor), GM-CSF (recombinant human granulocyte-macrophage colony-stimulating factor), multi-CSF (multiple colony-stimulating factor, also known as IL-3), EPO (erythropoietin), TPO, SCF, FlT3L; further preferably, the colony stimulating factor of the seventh medium comprises TPO.

[0100] Preferably, the interleukin of the seventh medium comprises any one of: IL-1, IL-2, IL-6, IL-7, IL-11, IL-15, IL-18, IL-21, IL-27; further preferably, the interleukin of the seventh medium comprises IL-11.

[0101] Preferably, the concentration of the colony stimulating factor in the seventh medium is 25-100 ng / mL; further 25-50 ng / mL; still further 50 ng / mL.

[0102] Preferably, the concentration of the interleukin in the seventh medium is 5-25 ng / mL; further 5-15 ng / mL; still further 10 ng / mL.

[0103] Preferably, the basal medium of the seventh medium comprises at least one of: IMDM (Iscove's Modified Dulbecco's Medium), DMEM medium, Ham's F-12 medium, STEMdiff TM APEL TM 2 medium, SFM medium; further preferably, the basal medium of the seventh medium comprises SFM medium.

[0104] Preferably, the SFM medium comprises: DMEM / F12, IMDM, BSA, CD lipid concentrate, GlutaMAX supplement (a substitute for L-glutamine), ITS, L-Ascorbic acid 2-phosphate, and 1-thioglycreol.

[0105] In the present application, the seventh culture medium is used for megakaryocyte to produce platelet.

[0106] Preferably, the megakaryocyte is mature megakaryocyte.

[0107] In a third aspect of the present application, there is provided use of the kit of the first aspect of the present application or the second aspect of the present application.

[0108] The kit of the first aspect of the present application is used in any one of (1) to (8);

[0109] (1) to prepare mesendoderm cells; (2) to prepare hemogenic endothelial cells; (3) to prepare megakaryoerythroid progenitor cells; (4) to prepare megakaryocytes; (5) to prepare a product for inducing stem cells to differentiate into mesendoderm cells; (6) to prepare a product for inducing stem cells to differentiate into hemogenic endothelial cells; (7) to prepare a product for inducing stem cells to differentiate into megakaryoerythroid progenitor cells; (8) to prepare a product for inducing stem cells to differentiate into megakaryocytes.

[0110] The kit of the second aspect of the present application is used in any one of (1) to (10);

[0111] (1) to prepare mesendoderm cells; (2) to prepare hemogenic endothelial cells; (3) to prepare megakaryoerythroid progenitor cells; (4) to prepare megakaryocytes; (5) to prepare a product for inducing stem cells to differentiate into mesendoderm cells; (6) to prepare a product for inducing stem cells to differentiate into hemogenic endothelial cells; (7) to prepare a product for inducing stem cells to differentiate into megakaryoerythroid progenitor cells; (8) to prepare a product for inducing stem cells to differentiate into megakaryocytes; (9) to prepare platelets; (10) to prepare a product for inducing stem cells to differentiate into platelets.

[0112] Preferably, the megakaryocyte is mature megakaryocyte.

[0113] Preferably, the stem cell is a stem cell with pluripotency.

[0114] Preferably, the stem cell with pluripotency comprises at least one of embryonic stem cell, parthenogenetic stem cell, induced pluripotent stem cell, mesenchymal stem cell, adipose stem cell, cord blood stem cell; further preferably, the stem cell with pluripotency comprises induced pluripotent stem cell.

[0115] Preferably, the stem cell is derived from mammal; further derived from primate, and more further derived from human.

[0116] Preferably, the embryonic stem cell is commercialized human embryonic stem cell line.

[0117] Preferably, the embryonic stem cell is a stem cell isolated or obtained from a human embryo that has not developed in vivo for 14 days or less from fertilization.

[0118] In a fourth aspect of the present application, there is provided a method for inducing differentiation of stem cells into megakaryocytes, comprising the step of using the kit of the first or second aspect of the present application.

[0119] Preferably, the method comprises the following steps:

[0120] (1) culturing the stem cells into mesendoderm cells to obtain mesendoderm cells;

[0121] (2) culturing the mesendoderm cells into hemogenic endothelium to obtain hemogenic endothelium cells;

[0122] (3) culturing the hemogenic endothelium cells in a fourth medium to obtain megakaryocyte-erythroid progenitor cells;

[0123] (4) culturing the megakaryocyte-erythroid progenitor cells into megakaryocytes to obtain megakaryocytes.

[0124] Preferably, the culturing in step (1) is performed in the first medium of the kit of the first or second aspect of the present application.

[0125] Preferably, the culturing in step (2) is performed in the second and third media of the kit of the first or second aspect of the present application; further preferably, the culturing in step (2) is performed in the second and third media of the kit of the first or second aspect of the present application in sequence.

[0126] Preferably, the culturing in step (4) is performed in the sixth medium of the kit of the first or second aspect of the present application.

[0127] Preferably, the culturing in step (1) is performed for 24 to 96 hours; further for 48 to 72 hours; and more further for 48 hours.

[0128] Preferably, the culturing in step (2) is performed for 48 to 120 hours; further for 72 to 120 hours; and more further for 96 hours.

[0129] Preferably, the culturing in step (2) in the second medium of the kit of the first or second aspect of the present application is performed for 24 to 72 hours; further for 24 to 48 hours; and more further for 24 hours.

[0130] Preferably, the culturing time of the third medium in step (2) in the kit of the first aspect or the second aspect of the present application is 48-120 hours; further 72-96 hours; more further 72 hours.

[0131] Preferably, the third medium is replaced every 48 hours during the culturing of the third medium in the kit of the first aspect or the second aspect of the present application.

[0132] Preferably, the culturing time of step (3) is 48-120 hours; further 72-96 hours; more further 72 hours.

[0133] Preferably, the culturing time of step (4) is 96-192 hours; further 120-168 hours; more further 144 hours.

[0134] Preferably, the sixth medium is replaced every 48 hours during the culturing of step (4).

[0135] Preferably, the culturing conditions of steps (1), (2), (3) are 32-38°C, 4-6% CO2, 4-6% O2.

[0136] Preferably, the culturing conditions of step (4) are 32-38°C, 4-6% CO2, 15-25% O2.

[0137] Preferably, between steps (3), (4) further comprises a step of culturing megakaryocytic-erythroid progenitor cells.

[0138] Preferably, the culturing of megakaryocytic-erythroid progenitor cells is performed in the fifth medium in the kit of the first aspect or the second aspect of the present application.

[0139] Preferably, the culturing conditions are 32-38°C, 4-6% CO2, 4-6% O2.

[0140] Preferably, the stem cells are stem cells with multi-differentiation potential.

[0141] Preferably, the stem cells with multi-differentiation potential comprise at least one of embryonic stem cells, parthenogenetic stem cells, induced pluripotent stem cells, mesenchymal stem cells, adipose stem cells, cord blood stem cells; further preferably, the stem cells with multi-differentiation potential comprise induced pluripotent stem cells.

[0142] Preferably, the stem cells are derived from mammals; further from primates, more further from humans.

[0143] Preferably, the embryonic stem cells are commercialized human embryonic stem cell lines.

[0144] Preferably, the embryonic stem cell is a stem cell isolated or obtained from a human embryo less than 14 days old that has not developed in vivo.

[0145] A method of inducing stem cells to differentiate into platelets, comprising the steps of using the kit of the second aspect of the present application.

[0146] Preferably, the method comprises the steps of a method of inducing stem cells to differentiate into megakaryocytes.

[0147] Preferably, the method further comprises: (5) culturing the megakaryocytes in the seventh medium in the kit of the second aspect of the present application to obtain platelets.

[0148] Preferably, the culturing in step (5) is for 144-288 hours; further for 192-240 hours; and more further for 192 hours.

[0149] Preferably, the seventh medium is replaced every 48 hours during the culturing in step (5).

[0150] Preferably, the culturing in step (5) is at 32-38°C, 4-6% CO2, and 15-25% O2.

[0151] In the fifth aspect of the present application, a megakaryocyte is provided, which is prepared by the method of inducing stem cells to differentiate into megakaryocytes of the third aspect of the present application.

[0152] Preferably, the megakaryocyte comprises more than 50% of 2N polyploid cells; further 50-54%.

[0153] Preferably, the megakaryocyte comprises more than 30% of 4N polyploid cells; further 30-32.3%.

[0154] Preferably, the megakaryocyte comprises more than 4% of 8N polyploid cells; further 4-4.9%.

[0155] Preferably, the megakaryocyte comprises more than 1% of polyploid cells with more than 16N; further 1-1.39%.

[0156] Preferably, the megakaryocyte comprises more than 90% of CD41a + CD42a + cells; further more than 95%; and more further 95-95.5%.

[0157] Preferably, the megakaryocyte comprises more than 90% of CD41a + CD42b +The cell content is greater than 85%; further greater than 90%; still further 90-90.5%.

[0158] In a fifth aspect, the present application provides a platelet prepared by the method of inducing stem cells to differentiate into platelets according to the third aspect of the present application.

[0159] Preferably, the CD61 content in the platelets is greater than 90%; further greater than 95%; still further 95-96.2%. + The platelet content is greater than 90%; further greater than 95%; still further 95-96.2%.

[0160] Preferably, the content of platelets expressing CD62p under the co-stimulation of ADP and TRAP-6 in the platelets is greater than 90%; further greater than 95%; still further 95-96.5%.

[0161] Preferably, the content of platelets binding to PAC-1 under the co-stimulation of ADP and TRAP-6 in the platelets is greater than 40%; further greater than 45%; still further 45-51%.

[0162] In a sixth aspect, the present application provides a pharmaceutical composition comprising the platelets according to the fifth aspect of the present application.

[0163] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0164] Preferably, the pharmaceutical composition is a liquid preparation.

[0165] Preferably, the pharmaceutical composition is an intravenous injection reagent.

[0166] Preferably, the pharmaceutically acceptable carrier includes, but is not limited to, saline, buffer, glucose, water, DMSO, and combinations thereof.

[0167] In a seventh aspect, the present application provides use of the platelets according to the fifth aspect of the present application and / or the pharmaceutical composition according to the sixth aspect of the present application in the preparation of a medicament.

[0168] Preferably, the medicament is for treating and / or preventing at least one of the following diseases: anemia, thrombocytopenia, acute leukemia.

[0169] Preferably, a method for treating a disease comprises the step of administering the platelets according to the fifth aspect of the present application and / or the pharmaceutical composition according to the sixth aspect of the present application to a subject.

[0170] Preferably, the subject is a mammal, more preferably a primate, still more preferably a human.

[0171] Preferably, the administration site is a vein of the subject.

[0172] Preferably, the disease comprises at least one of anemia, thrombocytopenia, acute leukemia.

[0173] The present application has the following beneficial effects:

[0174] The present application provides a kit for inducing stem cells to differentiate into megakaryocytes and / or platelets, which can induce stem cells to differentiate into megakaryocytes and / or platelets without introducing exogenous cells and exogenous genes, and can highly simulate the in vivo generation process of megakaryocytes and platelets, and can induce stem cells to differentiate into mesendoderm cells, hemogenic endothelial cells, and megakaryocyte-erythroid progenitor cells in sequence, and the megakaryocyte-erythroid progenitor cells further develop into megakaryocytes with multiploid characteristics, and finally produce stem cell-derived platelets with similar functionality to natural platelets, and the stem cells have the potential for unlimited proliferation, and can be massively expanded in vitro, which also provides an unlimited cell source for in vitro preparation of platelets. BRIEF DESCRIPTION OF DRAWINGS

[0175] Figure 1 is a schematic diagram of inducing human embryonic stem cells to differentiate into platelets.

[0176] Figure 2 is a schematic diagram of inducing human embryonic stem cells to differentiate into early hematopoietic cells (hemogenic endothelial cells) and a cell detection result diagram: wherein, A is a schematic diagram of inducing human embryonic stem cells to differentiate into hemogenic endothelial cells; B is a cell morphological change diagram of inducing human embryonic stem cells to differentiate into hemogenic endothelial cells (scale bar = 100 μm); C is a RT-qPCR detection result diagram of Brachyury, KDR, ERG and TIE1 in the process of inducing human embryonic stem cells to differentiate into hemogenic endothelial cells; D is a flow cytometry detection result diagram of hemogenic endothelial markers in the process of inducing human embryonic stem cells to differentiate into hemogenic endothelial cells; E is an immunofluorescence staining result diagram of CD43 of cell spheres on the sixth day of inducing human embryonic stem cells to differentiate into hemogenic endothelial cells (scale bar = 100 μm); * indicates: p < 0.05 compared with day 0; *** indicates: p < 0.001 compared with day 0; **** indicates: p < 0.0001 compared with day 0; ns indicates: p > 0.05 compared with day 0. + CD34 + hemogenic endothelial marker flow cytometry detection result diagram; E is an immunofluorescence staining result diagram of CD43 of cell spheres on the sixth day of inducing human embryonic stem cells to differentiate into hemogenic endothelial cells (scale bar = 100 μm); * indicates: p < 0.05 compared with day 0; *** indicates: p < 0.001 compared with day 0; **** indicates: p < 0.0001 compared with day 0; ns indicates: p > 0.05 compared with day 0.

[0177] Figure 3is a schematic diagram of inducing early hematopoietic cells (hemogenic endothelial cells) to differentiate into megakaryocytic-erythroid progenitor cells and a cell detection result diagram: wherein, A is a schematic diagram of inducing hemogenic endothelial cells to differentiate into megakaryocytic-erythroid progenitor cells; B is a cell morphological change diagram of inducing hemogenic endothelial cells to differentiate into megakaryocytic-erythroid progenitor cells (scale = 100 μm); C is a cell flow detection result diagram of CD43 in the process of inducing hemogenic endothelial cells to differentiate into megakaryocytic-erythroid progenitor cells; D is a cell flow detection result diagram of CD41a and CD235a in the process of inducing hemogenic endothelial cells to differentiate into megakaryocytic-erythroid progenitor cells.

[0178] Figure 4 is a schematic diagram of maintaining culture of megakaryocytic-erythroid progenitor cell spheres and a cell sphere detection result diagram: wherein, A is a schematic diagram of maintaining culture of megakaryocytic-erythroid progenitor cell spheres; B is a cell morphological change diagram in the process of maintaining culture of megakaryocytic-erythroid progenitor cell spheres (scale = 100 μm); C is a comparison result diagram of the number of continuously produced suspended cells in the process of maintaining culture of megakaryocytic-erythroid progenitor cell spheres; D is a cell flow detection result diagram of cell sphere CD31 and CD34, CD43, and CD41a and CD235a in the process of maintaining culture of megakaryocytic-erythroid progenitor cell spheres.

[0179] Figure 5 is a suspended cell detection result diagram in the process of maintaining culture of megakaryocytic-erythroid progenitor cell spheres: wherein, A is a cell flow detection result diagram of suspended cell CD43, CD41a and CD235a produced in the process of maintaining culture of megakaryocytic-erythroid progenitor cell spheres; B is a CD41a + suspended cell proportion statistical result diagram.

[0180] Figure 6 is a schematic diagram of inducing megakaryocytic-erythroid progenitor cells to differentiate into mature megakaryocytes and a cell detection result diagram: wherein, A is a schematic diagram of inducing megakaryocytic-erythroid progenitor cells to differentiate into mature megakaryocytes; B is a cell morphological diagram of inducing megakaryocytic-erythroid progenitor cells to differentiate into mature megakaryocytes (scale = 100 μm); C is a cell flow detection result diagram of CD41a and CD235a in the process of inducing megakaryocytic-erythroid progenitor cells to differentiate into mature megakaryocytes; D is a cell flow detection result diagram of CD41a and CD42a in the process of inducing megakaryocytic-erythroid progenitor cells to differentiate into mature megakaryocytes; E is a cell flow detection result diagram of CD41a and CD42b in the process of inducing megakaryocytic-erythroid progenitor cells to differentiate into mature megakaryocytes.

[0181] Figure 7Figure 1 is a cell detection result chart of the megakaryocytes on the 15th day of differentiation: A is a multiploid detection result chart of the megakaryocytes on the 15th day of differentiation; B is a Wright-Giemsa staining result chart of the megakaryocytes on the 15th day of differentiation (scale = 100 μm); C is an immunofluorescence staining result chart of CD62p of the megakaryocytes on the 15th day of differentiation (scale = 100 μm).

[0182] Figure 8 Figure 2 is a schematic diagram and cell detection result chart of the stage of platelet production by mature megakaryocytes: A is a schematic diagram of the stage of platelet production by mature megakaryocytes; B is a cell flow detection result chart of CD61 of the stage of platelet production by mature megakaryocytes; C is a cell flow detection result chart of CD42b and CD62p and CD42b and PAC-1 of the produced platelets before and after stimulation.

[0183] Figure 9 Figure 3 is a result chart of the body weight change, coagulation function and hemostatic function of the thrombocytopenia model mice: A is a result chart of the body weight change of the thrombocytopenia model mice; B is a result chart of the coagulation function of the thrombocytopenia model mice; C is a result chart of the hemostatic function of the thrombocytopenia model mice; ** indicates: p<0.01; **** indicates: p<0.0001; ns indicates: p>0.05.

[0184] Figure 10 Figure 4 is a result chart of the coagulation function, hemostatic function and proportion change of human CD61+ cells in the peripheral blood of the thrombocytopenia model mice after platelet infusion treatment: A is a detection result chart of the coagulation function of the thrombocytopenia model mice after platelet infusion treatment; B is a detection result chart of the hemostatic function of the thrombocytopenia model mice after platelet infusion treatment; C is a result chart of the proportion change of human CD61+ cells in the peripheral blood of the thrombocytopenia model mice after platelet infusion treatment; * indicates: p<0.05; ** indicates: p<0.01; *** indicates: p<0.001; ns indicates: p>0.05.

[0185] Figure 11 Figure 5 is a chart showing the effect of the culture medium on the differentiation efficiency of early hematopoietic cells (hemogenic endothelial cells) into megakaryocytic-erythroid progenitor cells: A is a schematic diagram of the differentiation of early hematopoietic cells (hemogenic endothelial cells) into megakaryocytic-erythroid progenitor cells; B is a cell flow detection result chart of the CD43, CD41a and CD235a markers of the cell spheres in the process of differentiation of early hematopoietic cells (hemogenic endothelial cells) into megakaryocytic-erythroid progenitor cells; C is a cell flow detection result chart of the CD43, CD41a and CD235a markers of the suspension cells produced in the process of differentiation of early hematopoietic cells (hemogenic endothelial cells) into megakaryocytic-erythroid progenitor cells.

[0186] Figure 12Figure is a diagram showing the effect of the medium on megakaryocyte-erythroid progenitor cell maintenance, wherein A is a schematic diagram of megakaryocyte-erythroid progenitor cell maintenance culture; B is a cell flow detection result diagram of CD43, CD41a and CD235a markers of cell spheres during the process of megakaryocyte-erythroid progenitor cell maintenance culture; C is a cell flow detection result diagram of CD43, CD41a and CD235a markers of suspension cells produced during the process of megakaryocyte-erythroid progenitor cell maintenance culture. DETAILED DESCRIPTION

[0187] The present application will be further described in details by specific examples.

[0188] It should be understood that the examples are only used for illustrating the present application but not for limiting the scope of the present application.

[0189] Definitions

[0190] The term "stem cell" refers to a cell that can self-replicate and has pluripotency or multipotency. Generally, stem cells can regenerate injured tissues. The stem cell herein can be, but is not limited to, an embryonic stem (ES) cell, an induced pluripotent stem cell or a tissue stem cell (also referred to as a tissue-specific stem cell or a somatic stem cell).

[0191] An "embryonic stem (ES) cell" is a pluripotent stem cell derived from an early embryo. ES cells were first established in 1981, and since 1989, they have also been applied to the production of knockout mice. In 1998, human ES cells were established, and they are now becoming available for regenerative medicine.

[0192] Unlike ES cells, tissue stem cells have limited differentiation potential. Tissue stem cells exist in specific locations in tissues and have undifferentiated intracellular structures. Therefore, the pluripotency of tissue stem cells is generally low. Tissue stem cells have a high nuclear / cytoplasmic ratio and have fewer intracellular organelles. Most tissue stem cells have low pluripotency, long cell cycles and the ability to proliferate over the life span of an individual. Tissue stem cells are divided into several categories based on the site from which the cells are derived, such as the skin system, the digestive system, the bone marrow system, the nervous system, etc. Tissue stem cells in the skin system include epidermal stem cells, hair follicle stem cells, etc. Tissue stem cells in the digestive system include pancreatic (common) stem cells, liver stem cells, etc. Tissue stem cells in the bone marrow system include hematopoietic stem cells, mesenchymal stem cells, etc. Tissue stem cells in the nervous system include neural stem cells, retinal stem cells, etc.

[0193] An "induced pluripotent stem cell" (often abbreviated iPS cell or iPSC) refers to a type of pluripotent stem cell artificially made from a non-pluripotent cell (typically a somatic cell) or a terminally differentiated cell (e.g. fibroblast, hematopoietic cell, muscle cell, neuron, epidermal cell, etc.) by the introduction of certain factors called reprogramming factors.

[0194] The term "differentiation" is a process by which a less specialized cell forms progeny of at least one more specialized new cell type.

[0195] The term "basal medium" belongs to a chemically defined medium, including but not limited to basal cell culture media such as IMDM (Iscove's Modified Dulbecco's Medium), DMEM medium, Ham's F-12 medium, STEMdiff TM APEL TM 2 medium, StemSpan TM SFEM II medium, etc.

[0196] The term "multipotential colony-stimulating factor" is also known as interleukin-3. It is mainly produced by activated Z cells or T cell clones. It contains 13 amino acid residues and can act as an immunomodulator, stimulating the proliferation and differentiation of pluripotent stem cells and various progenitor cells.

[0197] The term "SB431542" also includes SB431542 and salts thereof, in particular pharmaceutically acceptable salts.

[0198] The term "CHIR99021" includes CHIR99021 and salts thereof, in particular pharmaceutically acceptable salts.

[0199] The term "Y-27632" also includes Y-27632 and salts thereof, in particular pharmaceutically acceptable salts. A preferred pharmaceutically acceptable salt is Y-27632 2HCL.

[0200] The term "fibroblast growth factor" is a polypeptide secreted by the pituitary and hypothalamus, which can promote fibroblast mitosis, growth of mesodermal cells, and also stimulate vascular formation, playing a role in wound healing and limb regeneration, and has both acidic (pI 5.6) and basic (pI 9.6) forms, i.e. aFGF and bFGF.

[0201] The term "bone morphogenetic protein-4" (BMP4) has a regulatory effect on the proliferation and differentiation of various cells during the embryonic development period.

[0202] The experimental methods in the following examples, unless otherwise specified, were generally performed according to routine conditions, or according to the conditions recommended by the manufacturer. The materials, reagents, etc. used in the present examples, unless otherwise specified, were reagents and materials obtained from commercial channels.

[0203] Example 1 A kit for inducing differentiation of stem cells into platelets

[0204] A kit for inducing differentiation of stem cells into platelets, comprising: a first medium, a second medium, a third medium, a fourth medium, a fifth medium, a sixth medium, and a seventh medium;

[0205] The first medium is STEMdiff TM APEL TM 2 medium containing 10 μM Y27632, 10 ng / mL BMP4 (bone morphogenetic protein 4), 2 ng / mL Activin A, and 3 μM CHIR99021.

[0206] The second medium is STEMdiff TM APEL TM 2 medium containing 40 ng / mL VEGF (vascular endothelial growth factor).

[0207] The third medium is STEMdiff TM APEL TM 2 medium containing 40 ng / mL VEGF and 20 ng / mL bFGF (basic fibroblast growth factor).

[0208] The fourth medium is STEMdiff TM APEL TM 2 medium containing 50 ng / mL VEGF, 20 ng / mL bFGF, 3 U / mL EPO (erythropoietin), and 4 μM SB431542.

[0209] The fifth medium is STEMdiff TM APEL TM 2 medium containing 50 ng / mL SCF (stem cell growth factor), 100 ng / mL TPO (thrombopoietin), 20 ng / mL IL3 (multiple colony stimulating factor), 50 ng / mL IL6 (interleukin 6), and 20 ng / mL IL11 (interleukin 11).

[0210] The sixth medium is StemSpanTM SFEM II medium;

[0211] The seventh medium is SFM medium containing 50 ng / mL TPO and 10 ng / mL IL11, SFM medium is composed of 48 v / v% DMEM / F12, 48 v / v% IMDM, 0.5 v / v% BSA, 1 v / v% CD lipid concentrate, 2 mM GlutaMAX TM Additives (substitute for L-glutamine), 1% ITS, 50 μg / mL L-Ascorbic acid 2-phosphate and 437 μM 1-thioglycreol.

[0212] Example 2 A kit for inducing stem cells to differentiate into platelets

[0213] A kit for inducing stem cells to differentiate into platelets, comprising: a first medium, a second medium, a third medium, a fourth medium, a fifth medium, a sixth medium and a seventh medium;

[0214] The first medium is STEMdiff TM APEL TM 2 medium;

[0215] The second medium is STEMdiff TM APEL TM 2 medium;

[0216] The third medium is STEMdiff TM APEL TM 2 medium;

[0217] The fourth medium is STEMdiff TM APEL TM 2 medium;

[0218] The fifth medium is STEMdiff APEL medium containing 25 ng / mL SCF (stem cell growth factor), 50 ng / mL TPO (thrombopoietin), 10 ng / mL IL3 (interleukin 3), 50 ng / mL IL6, and 10 ng / mL IL11. TM APEL TM 2 medium;

[0219] The sixth medium is StemSpan SFEM II medium containing 10 ng / mL SCF, 50 ng / mL TPO, 10 ng / mL IL3, 10 ng / mL IL6, and 10 ng / mL IL11. TM SFEM II medium;

[0220] The seventh medium is SFM medium containing 25 ng / mL TPO and 5 ng / mL IL11, SFM medium consisting of 48% DMEM / F12, 48% IMDM, 0.5% BSA, 1% CD lipid concentrate, 2 mM GlutaMAX, 1% ITS, 50 μg / mL L-ascorbic phosphate, and 437 μM 1-thioglycreol.

[0221] Example 3 A kit for inducing differentiation of stem cells into platelets

[0222] A kit for inducing differentiation of stem cells into platelets, comprising: a first medium, a second medium, a third medium, a fourth medium, a fifth medium, a sixth medium, and a seventh medium;

[0223] The first medium is STEMdiff APEL medium containing 20 μΜ Y27632, 50 ng / mL BMP4 (bone morphogenetic protein 4), 20 ng / mL Activin A, and 5 μΜ CHIR99021. TM APEL TM 2 medium;

[0224] The second medium is STEMdiff APEL medium containing 100 ng / mL VEGF (vascular endothelial growth factor). TM APEL TM 2 medium;

[0225] The third medium is STEMdiff APEL medium containing 100 ng / mL VEGF and 50 ng / mL bFGF (basic fibroblast growth factor). TM APEL TM 2 medium;

[0226] The fourth medium is STEMdiff Hematopoietic Progenitor Cell Medium with 100 ng / mL VEGF, 50 ng / mL bFGF, 10 U / mL EPO (erythropoietin), and 20 μΜ SB431542 TM APEL TM 2 medium;

[0227] The fifth medium is STEMdiff Hematopoietic Progenitor Cell Medium with 100 ng / mL SCF (stem cell factor), 200 ng / mL TPO (thrombopoietin), 50 ng / mL IL3 (interleukin 3), 150 ng / mL IL6, and 50 ng / mL IL11 TM APEL TM 2 medium;

[0228] The sixth medium is StemSpan Hematopoietic Expansion Medium with 150 ng / mL SCF, 250 ng / mL TPO, 50 ng / mL IL3, 100 ng / mL IL6, and 40 ng / mL IL11 TM SFEM II medium;

[0229] The seventh medium is SFM medium with 100 ng / mL TPO and 25 ng / mL IL11, and the SFM medium is composed of 48% DMEM / F12, 48% IMDM, 0.5% BSA, 1% CD lipid concentrate, 2 mM GlutaMAX, 1% ITS, 50 μg / mL L-ascorbic phosphate, and 437 μΜ 1-thioglycreol.

[0230] Effect Example

[0231] Effect Example 1

[0232] A method for inducing differentiation of human embryonic stem cells into megakaryocytes and platelets, as shown in the schematic diagram Figure 1 , comprising the following steps:

[0233] (1) Differentiation of human embryonic stem cells into hematopoietic endothelial cells, as shown in the schematic diagram Figure 2 A in

[0234] 1) hESC culture, passage, and maintenance: The hESCs used in this example were cultured using mouse embryonic fibroblasts (MEF) as the feeder cells. One day before passage, six-well plates were coated with gelatin diluted to a final concentration of 0.1 wt% using 1×PBS buffer (the gelatin was stored at a concentration of 2% at 4°C and warmed to liquid state in a 37°C water bath before use). Two hours after placing the gelatin-coated six-well plates in a biosafety cabinet, MEF was inoculated, with 2-3 × 10⁶ MEF cells in each well. 5 For each MEF cell, add 2 mL of complete MEF medium (DMEM high-glucose medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin-gentamicin solution (triple antibiotics)) to each well and incubate at 37°C and 5% CO2 saturated humidity for one day. Select one well of healthy hESCs (purchased from WiCell Research Institute, USA; healthy hESCs: 70%-80% cell coverage after 5-6 days of passage, with tightly packed cell clones and clear edges, and no obvious differentiation). Discard the medium, wash once with 1×PBS, add 1 mL of 1 mg / mL type IV collagenase solution, and incubate at 37°C for 35-40 minutes. Observe the clonal morphology under a microscope to determine the appropriate digestion time. Digestion is complete when the hESC clone edges are bright or folded. Discard the collagenase solution, gently add 1 mL of DMEM / F12 medium to wash once, and discard the DMEM / F12 medium. Add 2 mL of hESC complete medium (DMEM / F12 medium containing 20% ​​serum substitute (KSR), 1 mM L-glutamine, 0.1 mM β-mercaptoethanol, 1% non-essential amino acids, and 10 ng / mL bFGF) to each well. Gently pipette the cells into centrifuge tubes, and gently separate the cell clusters into clumps of approximately 15 cells each. Seed the cell clumps at a ratio of 1:6 (one cell per well in a six-well plate) into six-well plates seeded with MEF. Incubate the cells at 37°C, 5% CO2, and saturated humidity, replacing the hESC complete medium daily.

[0235] 2) Mesoendodermal stage induction: When hESC cell clones reached approximately 80% confluence, the culture medium was discarded, and the cells were washed once with 1×PBS. 1 mL of GCDR was added, and the cells were incubated at 37°C for 5 minutes. The GCDR was then discarded, and 1 mL of the first culture medium from Example 1 was added. The cells were gently pipetted to form a single-cell suspension. The single-cell suspension was seeded into low-adhesion six-well plates, 1 million cells per well, and the first culture medium from Example 1 was added to 3 mL per well. The cells were statically cultured at 37°C, 5% CO2, and 5% O2 for 2 days to induce and produce mesoendodermal cell spheroids.

[0236] 3) Hemogenic endothelial stage induction: the medium of mesendoderm cell spheroids was replaced with the second medium in Example 1, and treated at 37°C, 5% CO2 and 5% O2 for one day, and then replaced with the third medium in Example 1 and cultured for one day; then replaced with fresh third medium in Example 1, and cultured at 37°C, 5% CO2 and 5% O2 for 2 days to obtain hemogenic endothelial cells.

[0237] (2) Early hematopoietic cell (hemogenic endothelial cell) to megakaryocyte-erythroid progenitor cell induction stage, the schematic diagram is shown in Figure 3 A of FIG. 1: the medium of early hematopoietic cells (hemogenic endothelial cells) obtained in step (1) was replaced with the fourth medium in Example 1, and the early hematopoietic cells (hemogenic endothelial cells) were further cultured at 37°C, 5% CO2 and 5% O2 for 3 days to induce the early hematopoietic cells (hemogenic endothelial cells) to differentiate into megakaryocyte-erythroid progenitor cells.

[0238] (3) Megakaryocyte-erythroid progenitor cell maintenance culture stage: part of the megakaryocyte-erythroid progenitor cells obtained in step (2) were taken for maintenance culture, and the schematic diagram is shown in Figure 4 A of FIG. 1, and the specific process is as follows: the suspension cells produced on the 9th day of differentiation were separated from the cell spheroids, and the cell spheroids were continuously cultured with the fifth medium in Example 1, and the newly produced suspension cells were separated every 3 days, and the fifth medium in Example 1 was replaced to continue the culture.

[0239] (4) Megakaryocyte-erythroid progenitor cell to mature megakaryocyte induction stage: part of the megakaryocyte-erythroid progenitor cells obtained in step (2) were taken to induce mature megakaryocytes, and the schematic diagram is shown in Figure 6 A of FIG. 1, and the specific process is as follows: the suspension cells produced on the 9th day of differentiation were collected, and the sixth medium in Example 1 was used for megakaryocyte lineage induction at 37°C, 5% CO2 and 20% O2 for 6 days, and the medium was replaced every two days.

[0240] (5) Mature megakaryocyte platelet production stage, the schematic diagram is shown in Figure 8 A of FIG. 1: the mature megakaryocytes differentiated to the 9th+6th day were collected, and the seventh medium in Example 1 was used for culture at 37°C, 5% CO2 and 20% O2 for 8 days, and the medium was replaced every two days to induce the mature megakaryocytes to produce platelets.

[0241] Detection of cells in step (1):

[0242] 1) Cell morphological changes: the cell spheroids were photographed every day for 6 days before differentiation, and the morphological changes of the cells were recorded; the results are shown in Figure 2As shown in Fig. 2B, after hESCs were digested into single cells or small cell clusters and inoculated into low-adhesion six-well plates and incubated for 1 day, regular and relatively uniform mesendoderm cell spheres with a diameter of 70-150 μm were formed; as the differentiation proceeded, the volume of the cell spheres became larger and larger, and when the differentiation reached the 6th day, the diameter of the cell spheres reached 300-400 μm; from the morphology, it can be seen that when the differentiation reached the 4th day, the interior of the cell spheres became transparent and hollow "capsular" structures began to appear, and on the 6th day, the "capsular" structures were more obvious.

[0243] 2) Cell RT-qPCR detection: the cell spheres were collected every two days within 6 days of differentiation, and the expression levels of mesoderm-specific genes (Brachyury), hematopoietic mesoderm and hematopoietic endothelium-specific genes (KDR) and early hematopoietic-specific genes (ERG and TIE1) were detected, and the results are shown in Fig. 2C. Figure 2 As shown in Fig. 2C, within 6 days of differentiation, the expression of the mesoderm-specific gene Brachyury increased significantly and reached a peak on the 2nd day of differentiation; as the differentiation proceeded, the expression of the hematopoietic mesoderm and hematopoietic endothelium-related gene KDR gradually increased, and the expression of the early hematopoietic-related genes ERG and TIE1 also gradually increased and reached a maximum on the 6th day of differentiation (the data of the 8th and 10th days in Fig. C are the data of megakaryocyte-erythroid progenitor cells).

[0244] 3) Cell flow detection: the cell spheres were collected every two days within 6 days of differentiation, and the expression levels of CD31 + CD34 + and hematopoietic endothelium-specific markers were detected, and the results are shown in Fig. 2D. Figure 2 As shown in Fig. 2D, the proportion of CD31 + endothelial cells gradually increased from the 2nd day of differentiation; the CD34 + hematopoietic cells began to appear on the 6th day of differentiation, and at this time, the proportion of CD31 + CD34 + hematopoietic endothelial cells first appeared, and the proportion was about 44%.

[0245] 4) Cell immunofluorescence staining: on the 6th day of differentiation, the cell spheres were collected to detect the expression of CD43 early myeloid-specific marker; and the results are shown in Fig. 2E. Figure 2 As shown in Fig. 2E, on the 6th day of differentiation, CD43 + early myeloid hematopoietic cells appeared in the cell spheres. The above results show that under the above culture medium combined with a low-oxygen condition, hESCs can be directed to induce early hematopoietic cells (hematopoietic endothelial cells) on the 6th day of differentiation.

[0246] Detection of the cells in step (2):

[0247] 1) Cell morphological changes: Cell spheres were photographed to record the morphological changes of the cells. The results are shown in Fig. 1A. Figure 3 Fig. 1B: Starting from the 8th day of differentiation, bright round individual cells or cell clusters began to appear on the originally smooth surface of the cell spheres; on the 9th day of differentiation, a large number of individual cells or cells loosely gathered together were observed in the culture medium.

[0248] 2) Cell flow cytometry: Cell spheres and generated suspension cells on the 6th and 9th day of differentiation were collected respectively to detect the expression levels of CD43, CD41a and CD235a. The results are shown in Fig. 2A and 2B. Figure 3 Fig. 2C and 2D: With the progress of differentiation, the expression of CD43 + The proportion of early myeloid hematopoietic cells increased (from 3.56% to 11.1%), and the proportion of CD235a + CD41a + The proportion of megakaryocytic erythroid progenitor cells also increased (from 0.095% to 4.14%); compared with cell spheres, the suspension cells collected on the 9th day of differentiation expressed a high proportion of CD43 + early myeloid hematopoietic markers (up to 97.7%), CD235a + CD41a + Megakaryocytic erythroid progenitor cells reached 76.7%, and in addition, about 10.6% of the cells expressed CD235a - CD41a + Megakaryocytic lineage markers. The above results show that under this differentiation system, early hematopoietic cell (hemogenic endothelial cell) spheres can be induced to differentiate into megakaryocytic erythroid progenitor cells.

[0249] Detection of cells in step (3):

[0250] 1) Cell morphological changes and cell number statistics: Cell spheres were photographed to record the morphological changes of the cells, and suspension cells were collected every 3 days to statistically analyze the cell number. The results are shown in Fig. 3A. Figure 4 Fig. 3B and 3C: Within the 9th to 21st day of differentiation, cell spheres could still continuously generate suspension cells. With the continuous shedding of suspension cells from the surface of the cell spheres, the volume of the cell spheres gradually decreased; in terms of morphology, within the first 18 days of differentiation, the cell spheres still maintained a relatively regular spherical shape, and when the differentiation reached the 21st day, some vacuole-like structures began to appear on the surface of the cell spheres Figure 4 Fig. 3B and 3C: Within the 9th to 21st day of differentiation, cell spheres could still continuously generate suspension cells. With the continuous shedding of suspension cells from the surface of the cell spheres, the volume of the cell spheres gradually decreased; in terms of morphology, within the first 18 days of differentiation, the cell spheres still maintained a relatively regular spherical shape, and when the differentiation reached the 21st day, some vacuole-like structures began to appear on the surface of the cell spheres Figure 4The number of floating cells produced within the first 18 days of differentiation increased, reaching a maximum at day 18, and then gradually decreased as the culture progressed. At day 21 of differentiation, the number of floating cells, although less than at day 18, was still relatively high compared to day 9. These results show that the megakaryocytic erythroid progenitor cell spheres can still be maintained in culture and continue to produce floating cells under this culture system.

[0251] 2) Cell flow cytometry: The megakaryocytic erythroid progenitor cell spheres and floating cells were collected every 3 days and the expression levels of CD31 and CD34, CD43 and CD41a and CD235a in the cell spheres and CD43, CD235a and CD41a in the floating cells were detected. The results are shown in Figure 4 、 5 The proportion of CD31 + CD34 + hematopoietic endothelial cells in the cell spheres was maintained at more than 30% throughout the culture period. At days 12, 15 and 18, a low proportion of CD43 + cells (2.46%, 6.36% and 8.33%, respectively) was detected. At day 21, the proportion of CD43 + cells increased to more than 50%. In addition, the proportion of CD235a + CD41a + cells was maintained at a low level (D in Figure 4 ), similar to the results in the previous stage (D in Figure 3 ). During differentiation, the proportion of CD43 + cells in the floating cells produced was as high as more than 95%. Although the proportion of CD235a + cells gradually decreased, the proportion of CD41a + cells was maintained at 60-80% (in Figure 5 ). In summary, under the induction of the fifth medium, the megakaryocytic erythroid progenitor cell spheres can be maintained in culture and contain more than 30% CD31 + CD34 + hematopoietic endothelial cells. At the same time, the floating cells produced can continuously differentiate into the CD41a + biased megakaryocyte lineage.

[0252] Detection of cells in step (4):

[0253] 1) Cell morphological changes: After 6 days of induction, the megakaryocytic erythroid progenitor cells produced larger diameter cells and filamentous pro-platelet structures (B in Figure 6 ).

[0254] 2) Cell flow cytometry: Cells were collected every two days during the 6 days of differentiation to detect CD41a. + CD235a + Megakaryocytic erythroid progenitor cells, CD41a + CD42a + and CD41a + CD42b + The expression levels of specific markers for mature megakaryocytes were as follows: Figure 6 As shown in C-E: From day 9+0 to day 9+2 of differentiation, CD235a + CD41a + The proportion of cells increased from 76.7% to 81.6%, and these cells were mainly composed of CD235a. - CD41a - Cells and CD235a + It originates from cell differentiation; from day 9+2 to day 9+6 of differentiation, CD235a... + CD41a + The proportion of cells gradually decreased, CD235a - CD41a + The proportion of cells gradually increases, CD235a + CD41a - Cell ratio and CD235a - CD41a - The proportion of cells did not change significantly, indicating that CD235a + CD41a + Cells gradually differentiate into CD41a + cell( Figure 6 (C). As megakaryocyte-induced differentiation progresses, the expression of specific markers CD41a and CD42a in differentiating cells gradually increases. Figure 6 (D). In the first two days (day 9+0 to day 9+2), CD41a + CD42a + Cell generation was rapid, with the proportion increasing 3.92-fold; subsequently, it increased slowly at a stable level, reaching over 95% by day 15 of differentiation. Furthermore, the expression of cell-specific markers CD41a and CD42b gradually increased with the progress of megakaryocytic differentiation. Figure 6 (E). In the first two days (day 9+0 to day 9+2), CD41a + CD42b +The proportion of cells increased most rapidly, increasing 4.79-fold; then it increased slowly, reaching over 90% by day 9+6 of differentiation. These results also indicate that during megakaryocyte development, the expression of CD42a and CD42b is synchronously upregulated, and over 95% of CD41a can be obtained by day 9+6 of differentiation. + CD42a + and over 90% of CD41a + CD42b + Mature megakaryocytes.

[0255] 3) Cell polyploidy detection: A key marker of mature megakaryocytes is the formation of polyploid cells through intranuclear mitosis. Propidine iodide is a chemical substance that can penetrate the cell membrane, embed itself in the nucleus of a double-stranded deoxyribonucleotide, and emit red fluorescence under specific laser excitation. The polyploidy ratio of cells was detected on day 9+6 of differentiation, and the results are as follows: Figure 7 As shown in Figure A, at day 9+6 of megakaryocyte differentiation, 54.0%, 32.3%, 4.92%, and 1.39% of cells, respectively, exhibited polyploid levels of 2N, 4N, 8N, and 16N or higher. These results indicate that treatment with the sixth culture medium produced mature megakaryocytes with polyploid characteristics.

[0256] 4) Wright-Giemsa staining: Wright-Giemsa staining working solution is a cell stain composed of two composite dyes, Swiss pigment and Giemsa dye. It can make the cytoplasm of cell smears appear orange or red, and the cell nucleus appear blue to purple. Wright-Giemsa staining is performed on cells at differentiation day 9+6 to identify the morphology of differentiated cells. The results are as follows: Figure 7 As shown in Figure B: the cytoplasm of mature megakaryocytes derived from hESCs is stained orange, and the nucleus is stained blue-purple. Observation reveals that the cells are relatively large, with a diameter of approximately 70–100 μm, numerous and large nuclei, and an irregular lobed shape. The cytoplasm is filled with numerous purplish-red microgranules of varying sizes, and the cell membrane has irregular, pseudopodia-like edges. These characteristics are consistent with those of mature megakaryocytes.

[0257] 5) Immunofluorescence staining: P-selectin (CD62p) is a glycoprotein on the surface of the α-granule membrane within platelets. When platelets are activated, CD62p is exposed on the platelet surface, participating in the coagulation process by mediating cell-cell adhesion. Furthermore, CD62p expression is also an important marker for platelet production by megakaryocytes. Cells were collected at day 9+6 of differentiation to detect the expression of CD62p, a platelet-specific marker. Results are as follows... Figure 7 As shown in Figure C, most differentiated megakaryocytes express CD62p, indicating their ability to produce platelets.

[0258] Detection of platelets and function in step (5):

[0259] 1) Flow detection: Collect platelets in the supernatant of the culture medium on the 9+6th and 9+14th day of differentiation (centrifuge the cells at 1000 rpm for 5 minutes, collect the supernatant of the culture medium, and centrifuge the platelets at 3000 g for 10 minutes), and detect the expression level of CD61. The results are shown in Fig. 2B: 72.1% of CD61 Figure 8 + Platelet production, when differentiated to the 9+14th day, CD61 + The proportion of platelets reached 96.2%. The results show that on the 9+6th day of differentiation, mature megakaryocytes have begun to produce platelets, and after 8 days of induction in the seventh medium, the purity of the produced platelets is higher.

[0260] 2) Detection of platelet function in vitro: Activation of platelets under the action of stimulants is an important indicator of the coagulation function of platelets. Before being activated, platelets are in a resting state and do not aggregate; when stimulated by substances such as collagen, ADP, and thrombin, activated platelets will expose P-selectin (CD62p) and at the same time have the ability to bind to PAC-1. Therefore, the in vitro function of differentiated platelets can be analyzed by activating them in vitro with stimulants such as ADP and detecting the expression of CD62p on their surface and their binding ability to PAC-1. Collect platelets in the supernatant of the cell culture medium on the 9+14th day of differentiation, and after 1 hour of stimulation of the platelets with ADP (final concentration of 100 μΜ) and TRAP-6 (final concentration of 40 μΜ) at room temperature, detect the expression of CD62p, CD42b on the surface of the platelets, and their binding ability to PAC-1. The results are shown in Fig. 2C: under the co-stimulation of ADP and TRAP-6, 96.5% of the platelets differentiated on the 9+14th day expressed CD62p, and 51% of the platelets showed binding ability to PAC-1. Figure 8

[0261] 3) Analysis of the function of platelets in vivo, which includes the following steps:

[0262] S1 Construction of a mouse model of thrombocytopenia

[0263] S11 Divide healthy 6-8 week old mice (purchased from Guangdong Medical Laboratory Animal Center) into 3 groups: 4 in the control group, injected with no solvent or drug; 4 in the negative control group, injected with an equal amount of normal saline (solvent) as the modeling group; 4 in the modeling group, injected with a cyclophosphamide solution; all mice were weighed and recorded on the day before injection of the drug (day 0);

[0264] ​​S12 The mice in the modeling group were subcutaneously injected with a cyclophosphamide solution (prepared with normal saline, concentration of 10 mg / mL) at a dose of 150 mg / kg for three consecutive days; the mice in the negative control group were subcutaneously injected with the same amount of normal saline as the mice in the modeling group for three consecutive days; the mice in each group were weighed and recorded for seven consecutive days;

[0265] S13 The coagulation ability (coagulation time and hemostasis time) of the mice in each group was detected on the fourth day of modeling;

[0266] S14 Mouse coagulation time detection method: mouse blood was collected by the orbital venous plexus method, a capillary tube was inserted into the inner canthus at an angle of 45 degrees to the alae nasi plane, and the capillary tube was rotated gently while pressure was applied, and the plasma flowed out of the capillary tube and dropped onto the cover glass, at which time the blood clotting time was recorded;

[0267] (A) Cover glass method: from 30 seconds, the blood on the cover glass was gently stirred with a needle tip every 15 seconds, and when filamentous coagulation appeared in the stirred blood, the time was recorded;

[0268] (B) Capillary tube method: from 30 seconds, the capillary tube was broken once every 30 seconds until filamentous coagulation appeared after the capillary tube was broken, and the time from the blood drop to the appearance of filamentous coagulation was recorded;

[0269] S15 Mouse hemostasis time detection method: a 23G needle was used to pierce the blood vessels 2 cm from the tip of the mouse tail to cause bleeding, and the tail wound was immediately placed in a transparent test tube filled with normal saline until the blood stopped flowing out, and the time from the blood vessel puncture to the hemostasis was recorded.

[0270] The body weight changes of the mice were recorded for 8 consecutive days, and the results are shown in Figure 9 A: the body weight of the normal mice and the mice injected with normal saline increased with the increase of the feeding days; in contrast, the body weight of the mice injected with cyclophosphamide gradually decreased within 3 days of injection, and reached the minimum on the 4th day, and the body weight of the mice began to recover after the injection of cyclophosphamide was stopped.

[0271] On the 4th day of modeling, the coagulation and hemostasis functions of the mice were detected, and the results are shown in Figure 9 B and C: compared with the normal mice, the blood clotting time of the model mice was significantly prolonged and the coagulation efficiency was significantly decreased, and there was no significant difference in the blood clotting time between the mice injected with normal saline and the normal mice; compared with the normal mice, the hemostasis time of the model mice was significantly longer after the blood vessels were pierced, and the hemostasis efficiency of the mice injected with normal saline was not significantly different from that of the normal mice.

[0272] The above results indicate that continuous injection of cyclophosphamide at a dose of 150 mg / kg into mice for three consecutive days effectively depletes platelets in the mice and leads to a significant decrease in coagulation and hemostasis functions in the model mice. Therefore, this method can effectively establish a thrombocytopenia model.

[0273] S2 platelet function detection and analysis

[0274] Following the method used in S1 to construct the thrombocytopenia model mouse, S21 constructed a control group of 4 mice (without injection of any solvent or drug) and a modeling group of 18 mice (injected with cyclophosphamide solution for three consecutive days).

[0275] On the fourth day of modeling, the mice in the modeling group were randomly divided into three groups: a placebo treatment group (injected with an equal volume of saline), a human platelet sample (donor-derived natural platelets) treatment group, and an hESC-differentiated platelet treatment group. The platelet injection dose in the human platelet sample treatment group and the hESC-differentiated platelet treatment group was the same, at 1×10⁻⁶. 8 Platelets were injected into mice via the tail vein.

[0276] S23 collected 20 μL of tail vein blood from mice at 0.5, 1, 2, 4, 6, 24 and 48 hours after platelet injection to detect the proportion of human platelets in the mice; the 20 μL of blood was diluted with 1 mL of 1×PBS buffer, labeled with AlexaFlour anti-human CD61 flow cytometry antibody, and detected using a BD FACS Celesta 12-color high-end flow cytometer;

[0277] Four hours after platelet injection, S24 tested the coagulation ability (clotting time and hemostasis time) of mice (using the same method as S1).

[0278] Experimental results are as follows Figure 10 As shown, on day 4 of thrombocytopenia modeling in mice, mice were treated with the same dose of hESC-differentiated platelets and donor-derived natural platelets, respectively. Microvenous blood samples were collected from the mice at 0.5, 1, 2, 4, 6, 24, and 48 hours after platelet injection to detect changes in exogenous platelet levels. The results are as follows... Figure 10 As shown in Figure C, the platelet count decreased significantly within the first hour after injection of platelets from both sources into mice, but stabilized after 2 hours.

[0279] The mice were injected with the same dose of hESC-differentiated platelets and donor-derived natural platelets respectively on the 4th day of modeling the thrombocytopenia model mice. The coagulation and hemostasis functions of the thrombocytopenia model mice were detected 4 hours after injection. As shown in Figure 10 Fig. 2A and B, compared with the placebo group (injected with normal saline for treatment), the blood clotting time of the mice injected with hESC-differentiated platelets and donor-derived natural platelets was significantly shortened, and there was no significant difference in the effect of the platelets from the two sources on the blood clotting efficiency of the mice, but slightly lower than that of normal mice; after treatment with the two kinds of exogenous platelets, the hemostasis efficiency of the mice was significantly improved, and the hemostasis effect of the two kinds of platelets was similar, and there was no significant difference compared with normal mice. The above results show that hESC-differentiated platelets can effectively restore the coagulation and hemostasis functions of thrombocytopenia model mice, and the effect is similar to that of natural platelets.

[0280] Effect of the medium of effect example 2 on the differentiation efficiency of hematopoietic endothelial cells into megakaryocyte-erythroid progenitor cells

[0281] S1 A method for inducing human embryonic stem cells to differentiate into megakaryocyte-erythroid progenitor cells, as shown in Figure 11 Fig. 1A, which is consistent with steps (1) and (2) of effect example 1.

[0282] S2 A method for inducing human embryonic stem cells to differentiate into megakaryocyte-erythroid progenitor cells, as shown in Figure 11 Fig. 1A, which is consistent with steps (1) and (2) of effect example 1, and the only difference is that the medium in step (2) is replaced by the fifth medium.

[0283] Comparison of the differentiation efficiency of hematopoietic endothelial cells into megakaryocyte-erythroid progenitor cells by the methods of S1 and S2, as shown in Figure 11 Fig. 2A and B. Figure 11 Fig. 2B is the cell flow detection results of the surface markers (CD43, CD41a and CD235a) of the cell spheres in the differentiation stage, and it can be seen that after 3 days of induction in the fourth medium or the fifth medium, 11.1% or 15.5% of the hematopoietic endothelial cell spheres produced CD43 + cells, respectively, and 4.14% or 5.95% of the hematopoietic endothelial cell spheres produced CD41a + CD235a + cells, respectively. Figure 11 Fig. 2C is the cell flow detection results of the surface markers (CD43, CD41a and CD235a) of the suspension cells produced in the differentiation stage, and it can be seen that after 3 days of culture in the fourth medium or the fifth medium, the produced suspension cells all highly expressed CD43 (more than 95%); under the induction of the fourth medium, the produced suspension cells had 0.1% CD235a + CD41a +Cells and CD235a - CD41a - The cell proportions were 76.7% and 6.76%, respectively; while under the induction of the fifth medium, the resulting suspension cells contained CD235a. + CD41a + Cells and CD235a - CD41a - The cell proportions were 62.7% (1.2 times lower than in the fourth medium) and 25.2% (3.5 times higher than in the fourth medium), respectively. Since megakaryocytes mainly exist as single, independent cells, the detection results of suspended cells are of greater interest and used as the judgment criterion. Based on the cell flow cytometry results, the S1 method, using the fourth medium, is more conducive to the production of megakaryocyte erythroid progenitors.

[0284] Effect of culture medium on the maintenance of megakaryocyte erythroid progenitor cells (Example 3)

[0285] S1 A method for inducing human embryonic stem cells to differentiate into megakaryotic erythroid progenitor cells, as illustrated in the diagram. Figure 12 As shown in Figure A, the steps (1), (2), and (3) are consistent with those in Example 1.

[0286] S2 A method for inducing human embryonic stem cells to differentiate into megakaryotic erythroid progenitor cells, as illustrated in the diagram. Figure 12 As shown in Figure A, the steps (1), (2), and (3) are the same as those in Example 1, except that the culture medium in step (3) is replaced with the fourth culture medium.

[0287] The effects of S1 and S2 methods on the maintenance of megakaryocyte erythroid progenitors were compared, and the results are as follows: Figure 12 As shown: Figure 12 The results of flow cytometry analysis of surface markers (CD43, CD41a, and CD235a) in cell spheres during the culture stage are shown in section B. It can be seen that under the influence of the fourth or fifth culture medium, CD43 levels within the cell spheres increased significantly. + Cells and CD235a + CD41a + The proportion of megakaryocyte erythroid progenitor cells remained at a low level; during these 3 days of culture, cell spheroids were able to continuously produce suspension cells. Figure 12 The results of flow cytometry analysis of surface markers (CD43, CD41a, and CD235a) in suspension cells produced during this culture stage are shown in Figure C. It can be seen that suspension cells induced by either the fourth or fifth culture medium highly express the early myeloid-specific marker CD43; the fourth culture medium induces the production of CD43... +The proportion of the suspended cells is about 82.4%, which is obviously lower than that induced by the fifth medium (about 95%, 1.15 times higher than that of the fourth medium); under the induction of the fourth medium, the proportion of CD41a + cells is only about 57%, which is 1.54 times lower than that of the last stage (the suspended cells of the ninth day under the induction of the fourth medium); among them, the proportion of CD235a + cells is about 24.9%, which is 2.44 times lower than that of the fourth medium; under the induction of the fifth medium, the proportion of CD41a + cells is about 80.5%, which is higher than that of the fourth medium; and the proportion of CD235a + cells is about 10.2%, which is 2.44 times lower than that of the fourth medium; from the results of the cell flow analysis, the method of S1, i.e., using the fifth medium, is more conducive to the maintenance of the megakaryocytic erythroid progenitor cells. - - The proportions of CD41a + cells and CD235a + cells are 32.6% and 24.9% respectively, which indicates that after the fourth medium is continuously induced for 3 days, the efficiency of producing the suspended cells of the megakaryocytic erythroid progenitor cells is obviously worse than that of the last stage; under the induction of the fifth medium, the proportion of CD41a + cells is 80.5%, which is higher than that of the fourth medium of the present stage, and the proportion of CD235a + cells is close to that of the suspended cells of the ninth day under the induction of the fourth medium of the last stage; and at this time, the proportion of CD235a - cells is about 10.2%, which is 2.44 times lower than that of the fourth medium; from the results of the cell flow analysis, the method of S1, i.e., using the fifth medium, is more conducive to the maintenance of the megakaryocytic erythroid progenitor cells. - The proportions of CD41a

[0288] The above embodiments are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above embodiments, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application shall be equivalent replacement modes, which are all included in the protection scope of the present application.​

Claims

1. A kit comprising: a first culture medium, a second culture medium, a third culture medium, a fourth culture medium, and a sixth culture medium; The first culture medium was a STEMdiff medium supplemented with 5–15 μM Y-27632, 5–25 ng / mL BMP4, 1–4 μM CHIR-99021, and 1–5 ng / mL Activin A. TM APEL TM 2. Culture medium; The second culture medium is STEMdiff culture medium supplemented with 40–60 ng / mL vascular endothelial growth factor. TM APEL TM 2. Culture medium; The third culture medium is STEMdiff culture medium supplemented with 10–20 ng / mL basic fibroblast growth factor and 40–50 ng / mL vascular endothelial growth factor. TM APEL TM 2. Culture medium; The fourth culture medium is a STEMdiff medium supplemented with 1–5 U / mL LEPO, 20–30 ng / mL basic fibroblast growth factor, 40–60 ng / mL vascular endothelial growth factor, and 1–5 μM SSB431542. TM APEL TM 2. Culture medium; The sixth culture medium is StemSpan supplemented with 50–100 ng / mL LSCAF, 75–125 ng / mL LTPO, 20–30 ng / mL IL3, 40–60 ng / mL IL-6, and 20–30 ng / mL IL-11. TM SFEM II medium; The first culture medium is used to induce stem cells to differentiate into mesoendothelial cells; The second and third culture media are used to induce mesoendothelial cells to differentiate into hematopoietic endothelial cells; The fourth culture medium is used to induce hematopoietic endothelial cells to differentiate into megakaryotic erythroid progenitor cells; The sixth culture medium is used to induce megakaryocytes to differentiate into mature megakaryocytes.

2. The reagent kit according to claim 1, characterized in that, The kit also includes a fifth culture medium, which is a StemSpan culture medium containing at least one or more of the following: TM SFEM II medium: 85–350 ng / mL colony-stimulating factor, 60–200 ng / mL interleukin; the fifth medium is used for the maintenance culture of megakaryocyte erythroid progenitor cells.

3. The reagent kit according to claim 2, characterized in that, The colony-stimulating factor in the fifth culture medium includes at least two of the following: G-CSF, M-CSF, GM-CSF, IL-3, EPO, TPO, SCF, and FlT3L.

4. The reagent kit according to claim 2, characterized in that, The interleukins in the fifth culture medium include at least two of the following: IL-1, IL-2, IL-6, IL-7, IL-11, IL-15, IL-18, IL-21, and IL-27.

5. The reagent kit according to claim 1, characterized in that: The kit also includes: a seventh culture medium; the seventh culture medium is an SFM culture medium containing at least one or more of the following: 25–100 ng / mL colony-stimulating factor, 5–25 ng / mL interleukin; the seventh culture medium is used for megakaryocytes to produce platelets.

6. The reagent kit according to claim 5, characterized in that, The colony-stimulating factor in the seventh culture medium includes any one of G-CSF, M-CSF, GM-CSF, IL-3, EPO, TPO, SCF, and FlT3L.

7. The reagent kit according to claim 5, characterized in that, The interleukins in the seventh culture medium include any one of IL-1, IL-2, IL-6, IL-7, IL-11, IL-15, IL-18, IL-21, and IL-27.

8. The use of the kit described in any one of claims 5-7 in any one of (1) to (2); (1) Used to induce stem cells to differentiate into megakaryocytes; (2) Used to induce stem cells to differentiate into platelets.

9. A method for inducing stem cell differentiation into megakaryocytes using the kit according to claim 1, characterized in that, The method includes the following steps: (1) Using the first culture medium in claim 1, stem cells are cultured to differentiate into mesoendodermal cells for 48 to 72 hours to obtain mesoendodermal cells; (2) Using the second and third culture media in claim 1, mesoendothelial cells are cultured to differentiate into hematopoietic endothelial cells for 72 to 120 hours to obtain hematopoietic endothelial cells; (3) Hematopoietic endothelial cells are cultured in the fourth culture medium described in claim 1 for 72 to 96 hours to obtain megakaryocyte erythroid progenitor cells; (4) Using the sixth culture medium described in claim 1, megakaryocytes are cultured to differentiate into megakaryocytes for 120 to 168 hours to obtain megakaryocytes.

10. A method for inducing stem cell differentiation into platelets using the kit according to any one of claims 5 to 7, characterized in that, The method includes the following steps: (1) Using the first culture medium, stem cells were cultured to differentiate into mesoendodermal cells for 48–72 hours to obtain mesoendodermal cells; (2) Use the second and third culture media to culture mesoendothelial cells into hematopoietic endothelial cells for 72-120 hours to obtain hematopoietic endothelial cells; (3) Hematopoietic endothelial cells were cultured in the fourth culture medium for 72-96 hours to obtain megakaryocyte erythroid progenitor cells; (4) The sixth culture medium is used to culture megakaryocytes into megakaryocytes for 120-168 hours to obtain megakaryocytes; (5) Megakaryotic cells were cultured in the seventh culture medium for 192-240 hours to obtain platelets.

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